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Alkaline Phosphatase Activity in the Developing 
Slime Mold, Dictyostelium discoideum Raper 






June, 1955 


To the members of oy graduate committee. Dr a. J. H. Gregg, 

*** Griffith, A* E# Grobman, J. V* Slater and T* W. Stearns, 
sincere appreciation is expressed for their critical reading of 
this manuscript* 

To Dr. J. H. Gregg, chairman of my graduate committee, I 
especially express my gratitude for introducing me to Dictyostellum 
digcoldeua . Furthermore, it is to him that I am indebted for ac- 
quainting me with some of the micro-techniques used in this study. 



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Figures j P&ge 

I. Diagram of life Cycle of Dlctyostellum diacoideum . . 3 ? 

Ila. Photomicrograph of Vegetative Myxasoebae ...... 38 

Ilh. Drawing of Vegetative Myxaaoebae . 38 

III. rseudoplasmodium •Evolving 1 from Aggregation. 

Maes .......... 39 

IV. Early Pseudoplasraodium ............... 40 

V. Late Paeudoplasmodium .41 

VI. Very Early Pre-culmlnated Individual ........ 42 

VII. Early Pre-culminated Individual ..... 43 

VIII. Middle Pre-culminated Individual .......... 44 

IX. Late Pre-culminated Individual . 45 

X. Young Soroearp 46 

XI. Late Culminated Individual 47 


XII. Mature Soroearp 48 

XIII. Graph of Spectrophotoaetrlc Data .......... 49 


- 1 - 


During the last decade, the field of enzymology has g a * n»d 
increasing importance as a vehicle with which to gain insight into 
the general problems of differentiation and morphogenesis* Indica- 
tions of this are the many reviews and summaries which concern them- 
selves with the relationships of enzymes and embryogenesis (Barth 
and Barth, 195 * i Brachet, 1950* Needham, 19*2* Boell, 1948j Gustafson, 

1 95*)* A simultaneous rise in our knowledge of the importance of the 
roles of phosphorus and phosphorylated compounds in metabolic processes 
has also been evidenced within the last ten years* Their importance 
i® such, "•••that the most extraordinary achievement of some future 
investigator may well be the discovery of a metabolic system in which 
phosphorus is demonstrably not involved" (Glass, 1952)* In the light 
of these pronounced trends, it is plausible to pursue a problem which 
attempts to relate the two by embracing a study of an enzyme involved 
in phosphate metabolism and its possible relationship to the processes 
of differentiation and morphogenesis* An investigation of alkaline 
phosphatase activity in the developing slime mold, Dictvosteliim 
discoideum Raper, is such a study. 

The enzyme, alkaline phosphatase, has been classified as a 
hydrolytic enzyme, i*e«, its action is one of hydrolysis rather than 
of synthesis (Roche, 1950 )• It is activated in the presence of divalent 


cations, especially magnesium, and inhibition of activity is 
accomplished by sulfhydryl compounds and the arsenate ion* The 
optimal pH for maximtan activity is about 9.0-9.5. Substrate speci- 
ficity is limited to the orthophosphoric monoesters* 

The distribution of alkaline phosphatase in the animal king- 
dom is almost universal* According to Roche (1950), it has been 
found to exist in every animal cell except those of hyaline cartilage 
and those of the tunics of vascular vessels, while its presence in 
the plant kingdom is limited to the bacteria and yeasts* 

In terms of possible functional roles of the enzyme, the distri- 
bution of alkaline phosphatase within the individual organism has been 
classified into three main categories (Bradfield, 1950)* First, the 
enzyme is found in sites where active transfer of solutes across 
cell membranes is known to occur* The kidneys of many max isls, as 
well as those of other classes of vertebrates, are rich in alkaline 
phosphatase activity, especially in the region of the proximal con- 
voluted tubule. White and Schmitt (1926) have shown that it is at 
this level of the kidney that reabsorption of glucose occurs* Blood 
capillaries (Jacoby, 19*6) as well as placentae (Wislocki and Dempsey, 
19*6) have also been shown to have high degrees of alkaline phosphatase 

Secondly, a high correlation has been shown to exist between 
sites of pronounced alkaline phosphatase activity and areas where 
calcification processes are occurring, Robison (1923), Fell and 
Robison (I929), and Moog (19**) have demonstrated this enzyme in the 
bones of birds and mammals, while Engel and Furuta (19*2), and 
Bevelander and Johnson (1945) have demonstrated the activity of this 
enzyme in mammalian teeth. The bones, scales, and teeth of fish also 
exhibit high levels of alkaline phosphatase activity (Roche and 
Bullinger, 1939)* Evidently the enzyme acts in the role of concentrat- 
ing phosphate ions for the eventual deposition of calcium phosphate 
(Bradfield, 1950), 

In the third instance, alkaline phosphatase has been shown to 
be intimately associated with areas in which the metabolism of nucleic 
acids and proteins is occurring, Danielli and Catcheside (19*5), and 
Krugelis (19*6) have shown the existence of alkaline phosphatase activity 
in certain bands of the giant salivary chromosomes of Droanphijn . It is 
in these same bands that desoxyribonucleotides have also been shown to 
be present, Brachet (19*7) has demonstrated a positive correlation 
between the presence of the enzyme and high DNA turnover in the intestinal 
mucosa, testis, and spleen of the mouse. In addition, high alkaline 
phosphatase activity is found in the silk glands of spiders and cater- 
pillars, with the site of activity occurring between the nucleoproteins 

of the cytoplasm and the lumen of the gland (Bradfield, 1951). 

Alkaline phosphatase activity becomes more pronounced with increased 
d-uferentiation of embryonic tissue when protein synthesis occurs at 
the expense of yolk (Moog, 1944, 19461 Bracket, 19*6 j KrugeliB, 1947). 
Bradfield (19*6) and Jeener (19*7) have expressed the opinion that 
alkaline phosphatase is concerned with the synthesis of fibrous 
rather than globular proteins* 

V7ith regard to the experimental organism, the life cycle of 

D. disco id eun has been adequately described (Bonner, 1944j Raper, 

, ' • 

1935, 1940$ Raper and Fennell, 1952). For purposes of orientation, 
however, a resume of the life cycle is presented here. Individual 
myxamoebae (Figure I, 1) germinate from each of the encapsulated 
spores which are released from the sorogen (spore cap) of the mature 
sorocarp. The myxamoebae feed upon bacteria and grow in size as well 
as in nunbers. Upon completion of the vegetative stage, aggregation 
consaences and is characterized by a streaming of all myxamoebae 
toward a central focal point. It is at this focal point that all 
individual myxamoebae coalesce to form an integrated aggregation 
mass (Figure I, 2). The streaming phenomenon is thought to be initiat- 
ed by some chsootactic stimulus (Bonner, 194-7). Both syngamy and meiotic 
activity have been described during the aggregation stage (Wilson, 1953). 

As the aggregation mass increase*? in height, it topples over, 
and the tip comes in contact with the substrate (Figure I, 3), From 
this aggregation mass there evolves a nigra ting psettdoplssmodium 
(Figure I, A) which moves over the surface of the substrate# It has 
been demonstrated that the anterior one-third of the pseudoplaamodium 
is composed of cells (pre-stalk) which will eventually give rise to 
the stalk of the mature sorocarp* The remaining portion of the 
pseudoplaamodium is made up of cells (pre-spore) which will give rise 
to the spores of the mature individual* 

After a period of time determined by environmental conditions 
(Slifkin and Bonner, 1952), the pseudoplasmodium ceases to migrate 
and reorganizes itself (Figure I, 5). This individual, which is now 
in the pre-culmination stage, consists of pre-spore cells with an 
apex composed of pre-stalk cells* During the succeeding culmination 
stage (Figure I, 6), the pre-spore mass is raised above the substrate 
and is supported by the stalk. It is during this developmental stage 
that the transformation of the individual pre-spore myxamoebae into 
encapsulated spores takes place* Wilson (1953) has described the 
occurrence of mitotic activity during the time of spore formation* 

The raising of the spore mass into the air is continued until the 
sorogen is finally situated at the highest point of the supporting 
stalk (Figure I, 7)* This is the mature sorocarp* 

- 6 - 

It has been suggested upon numerous occasions (Bonner, 1944, 
19*7* Gregg, 1950| Raper and Fennell, 1952) that D. dlscoideum is 
ideally suited for the study of differentiation and morphogenesis, 
since it was thought that in this organism there was no overlap or 
"masking effect" of growth processes over those of morphogenesis* 
Wilson's work however seems to cast some doubt upon this concept* 
Bonner and Frascella (1952) maintain that, even though their observa- 
tions agree to some extent with those of Wilson, "differentiation is 
not dependent on mitoses*" 

It has been the purpose of this work to study the enzyme, 
alkaline phosphatase, in the slime mold, D, dlscoideum . from both 
qualitative and quantitative viewpoints* The qualitative aspect en- 
tailed the use of Oomori's method for the histochemical localization 
of the enzyme (1952) to demonstrate sites of alkaline phosphatase 
activity in successive stages of the developmental cycle* The second 
approach was quantitative in that speetrophotometric analyses of 
alkaline phosphatase activity were made in order to measure the degree 
of activity in the various stages of development. An attempt is made 
to correlate the data and observations derived from these methods with 
already existing biochemical data concerned with differentiation and 



Culture of Dlctyogtelium dlscoideura 

B. discoldema was grown In a two-raeabered culture on a nutrient 
agar medium. In addition to the spores from the mature sorocarps of 
D, diecoideum . the medium was inoculated with Escherichia coll . The 
nutrient agar medium was Bade according to Bonner (19^?) and consisted 
of the following components: 

A gar 



Distilled water to make 

10.00 gm. 

10.00 gm. 

20.00 gm. 
0.96 gm. 

1.45 gnu 

1000.00 ml. 

Stock cultures were maintained in test tube slants, while cultures 
from which organisms were harvested for experimentation were grown 
on media contained in Petri plates. 

Hlstocheaiical Localization of Alkaline Phosphatase 

The method of Goraori (1952) was used to demonstrate histo- 
chen&eally the sites of alkaline phosphatase occurrence. This method 
is essentially the same as was originally published by Goraori (1939) 
and Takamatsu (1939). The histoeheraieal localization of alkaline 
phosphatase is determined by a series of replacement reactions. The 
tissue is incubated in a buffered medium containing the substrate. 

— 8 — 

sodium glycerophosphate, The phosphate radical is cleared and com- 
bines with the calcium ion in the medium to form Ca^(P0^) 2 . The tissue 
is then i merged in e dilute cobalt salt solution, and calcium is re- 
placed by cobalt to form V/hen the tissue is exposed to the 

final solution, ammonium sulfide, the sulfide replaces the phosphate 
radical, and black precipitous CoS results. The final precipitate of 
CoS determines the localisation sites of alkaline phosphatase. 

Desired stages in the development of D. discoldeum were grown as 
already described, Fixation of the tissue was achieved by flooding the 
entire Petri plate with ice-cold 80$ ethanol. The flooded plate was 
then put in the refrigerator (5-10° C.) for one hour. After this period 
of time, the tissues were dehydrated by placing them in ice-cold absolute 
. ethanol for two hours. Cold temperatures were maintained throughout the 
fixation period so as not to denature the enzyme. After dehydration 
had been accomplished, the individual organisms were passed through two 
chloroform baths, approximately fifteen minutes in each bath. From the 
chloroform, the tissues were passed directly into melted Fisher Tissue- 
Mat (a.p. 50-52° C.). Oven temperature was maintained at no hi^aer 
than 55° C., since Goaori (1952) and Banielli (1953) have indicated 
that sustained high temperatures may destroy the enzymatic activity. 

When the Tissue-Mat had thoroughly impregnated the tissue (usually one 
to two hours), the organisms were oriented in the desired position, 
and the paraffin was allowed to solidify. 


All stages of development except the vegetative myxamoebae wore 
processed in this manner. The vegetative myxamoebae ware harvested 
in the manner described by Bonner (19*7), and a smear of these cells 
was made according to Guyer (1953)* The smear of myxamoebae was then 
fixed in 80^ ice-cold ethanol and subjected to the histochemical 
method of Gomori. 

All organisms, except the vegetative myxamoebae, were handled 
individually after the initial fixation with Sof, ethanol. Transfers 
of the tissues from one medium to another were made by means of 
watchmaker’s forceps, hair loops, or the tip of a very fine teasing 

All sections were cut at 10 micra* After mounting on slides, 
the serial sections ware incubated in the following medium at 37° C* 
for three hours* 

After incubation the slides were immersed in a 7$ solution of cobalt 
chloride for five minutes. Following a two minute rinse in circulating 
tap water, the serial sections were placed in an ammonium sulfide 
solution (ten drops of ammonium sulfide in a Coplin jar of distilled 

3^ sodium glycerophosphate 
Zf» calcium chloride 
1<$ magnesium sulfate 
Sodium barbital 
Distilled water to make 

10.0 ml. 

25.0 ml. 

10.0 dp. 
0.7 gm. 

50.0 ml. 

water) for five minutes. To completely wash away all traces of the 
reagents through which the tissue has been passed, the elides were 
washed in circulating tap water for at least ten minutes* The sections 
were then dehydrated and mounted in Piccolyte* Corresponding control 
slides were made in exactly the same manner with the exception that 
the substrate, sodium glyc ero phosphate t was emitted from the incubat- 
ing medium* 

■^.getrophotometric Analysis of Alkaline 
Phosphatase Activity 

Spectrophotometrie analyses of alkaline phosphatase activity 
were performed in essentially the Bame manner as was done by Krugelis, 
jgt &1* (1952) and Krugelis (1950)* Four stages in the developmental 
cycle of D* discoideim were chosen on which to do the analyses* These 
were the vegetative myxamoebae, migrating pseudoplasmodia, young soro- 
carps, and mature sorocarps* Harvesting of myxamoebae was done in the 
manner already described* The individual migrating pseudoplasmodia 
and young sorocarps were picked from the agar surface with the aid of 
a hair loop and transferred to the grinding surface of the homogeniser* 
The young sorocarp stage was defined as that stage of development in 
which the sorogen had been raised above the substrate but in which the 
individual was not as yet mature* Watchmaker’s forceps were used to 
harvest the nature sorocarps individually. 

In view of the fact that the pseudoplasmodium crawls over the 
same substrate upon which E, coli is feeding, several experiments 
were performed (Gregg, 195*) to determine whether E. coli was being 
harvested with the slime mold and contributing any enzymatic activity 
to that in the slime mold* D. diacoideum was allowed to develop to 
the point where aggregation masses were formed* Circular disce of 
agar upon which the aggregation masses were situated were then cut 
out with a large size cork borer* These discs were transferred to 
cut-outs of exactly the same size which were previously made in non- 
nutrient agar medium. The aggregation masses then developed into 
migrating pseudoplasmodia which migrated off the nutrient agar discs 
onto the non-nutrient agar substrate* It was believed that any E* 
soil which might have adhered to the pseudoplasmodium as it migrated 
from the nutrient agar would be lost as it migrated over the non- 
nutrient agar* Because no pronounced differences in final results 
were evident between experiments performed on individuals gathered 
from nutrient and non-nutrient agar, the former procedure was con- 

Organisms, harvested in the desired stage of development, were 
homogenized in a micro-homogenizer of the type described by Gregg, 
et ej* ( 195 4 ). Water was used as the extracting medium* The homo- 
genate was diluted to a volume of 1,0 ml, and thoroughly mixed. With 

12 - 

the use of micro-pipettes, aliquots of the homogenate were taken to 
determine tissue dry weight as well as alkaline phosphatase activity. 

Tissue dry weight determinations were made in the following 
manner i An aliquot of the homogenate was taken and transferred to a 
thin circular collodion membrane having the diameter of a No. 3 cork 
borer. Since the size of the membrane could not aceomodate more than 
20 /il of brei at one time, several 20 /il fractions were added on the 
membrane, with each fraction being dried before the succeeding fraction 
was added. After a sufficient amount of the homogenate had been added, 
the preparations were placed in a drying oven at 60° C. for a period 
of twelve hours. The tissue was weighed on a quartz helical balance 
having a range of from 1.0 to 1000 /ig. 

Two 0.3 ml. aliquots of the tissue brei were used for measuring 
alkaline phosphatase activity, and one 0*3 ml. aliquot was used as a 
corresponding control. The three aliquots were centrifuged to remove 
all cell debris and particulate matter* The supernatants, which 
served as the source of enzyme, were quantitatively transferred to three 
separate tubes. To each of these three tubes an equal volume of buffer- 
ed substrate was added and then thoroughly mixed* The buffered substrate 
was composed of the following components* 

Sodium glycerophosphate 
Sodium barbital 
Magnesium sulfate 

10 mg/ml H 2 O 
40 Eg/ml H 2 O 
2*5 mg/ml HgO 


The pH of the buffered substrate, 9*3* was measured by means of a 
Beckman pH meter* 

Immediately upon the addition of the buffered substrate to the 
enzyme extract, the reaction in the control tube was stopped with the 
addition of an equal amount (0.6 ml) of trichloroacetic acid (TCA). 
The experimental tubes, however, were allowed to incubate at room 
temperature for a period of three hours, and then the enzymatic reaction 
was also stopped with an equal amount of 2$% TCA* Following this, the 
three tubes, two experimentals and one control, were centrifuged to 
remove the precipitate* 

The supernatants were quantitatively transferred to a color re- 
action vessel, and a measure of the amount of phosphorus liberated 
fjrom the substrate by alkaline phosphatase (a measure of alkaline 
phosphatase activity) was made colorimetries lly. The determination 
was done according to the procedure of Kuttner and Cohen (1937), with 
modifications suggested by Krugelis (1950), This method is based on 
the reduction of phosphomolybdic acid by stannous chloride with a 
subsequent reading of the intensity of the formed color in a Beckman 
DU Spectrophotometer at 700 ja. 

To each of the supernatants was added each of the following 
reagents in the order listed* 

4 N sulfuric acid 
3 i» ammonium molybdate 
0.08/£ stannous chloride 

1*0 ml* 

1,0 ml, 
1,0 ml. 

After the addition of the last reagent, a period of fifteen minutes 
was allowed for the color to develop. The color intensity was then 

The amounts of phosphorus liberated were determined by comparing 
experimental extinction values with the extinction values of standard 
solutions containing known amounts of phosphorus* Results are ex- 
pressed in terms of P liberated during three hours of incubation at 
25 ° C, per pg dry weight of tissue. 

Several preliminary experiments were performed to determine the 
optimal pH, temperature, and substrate concentration. These experiments 
indicated that pH 9,3, 25 ° C. incubating temperature, and a substrate 
concentration of 10 ag/ml HgO were most favorable for optimal enzymatic 


- 15 - 


Hlatochecilcal Localization of Alkaline 

Hie results of the hietochemical localization of alkaline 
phosphatase appear in Figures IT through XXI, The photomicrograph 
of the vegetative myxamoebae (Figure Ila) has been supplemented with 
a drawing (Figure lib) made from a region of the same preparation in 
order to present more clearly the sites of enzymatic activity. Figures 
ill through XU represent sections of individual slime molds presented 
in successive stages of development. Control slides were made simul- 
taneously, but in no case was any darkening of the tissue obtained. 
Therefore, all dark areas on the experimental sections have been con- 
sidered sites of enzymatic activity. Photomicrographs of control 
slides are not presented because lack of contrast prevented the taking 
of clear pictures. 

Figures £nd lib . 

In the myxamoebae, sites of alkaline phosphatase activity can be 
seen in the cytoplasm immediately adjacent to the nuclear membrane. 

It is reasonably certain that no connection exists between these sites 
of activity, which occur several to a cell. Other sites of activity 
are noted in the cytoplasm, but they are not as pronounced in size as 
those next to the nuclear membrane. 


Figure HI* 

This is a section of a pseudoplasmodium evolving from an aggrega- 
tion mass. An area of pronounced alkaline phosphatase activity is 
evident in the most anterior extreme of the organism. The cells of 


the aggregation mass are compact toward the periphery, while the stream 
of cells forming the pseudoplaamodium exhibits a loose texture. The 
enzymatic activity in the cells of the entire organism, except at the 
most anterior end, ia comparable to the staining seen in the vegetative 

gfisa.3* • 

This section represents a relatively young migrating pseudo- 
plaamodium. The cells of the anterior end, i.e., the pointed end, 
are beginning to align themselves in a plane perpendicular to the 
longitudinal axis of the organism. This orientation becomes more 
evident in the late migrating pseudoplasmodium. Not all young migrating 
pseudoplasmodia, however, exhibit this cell orientation. This is in 
agreement with the findings of Bonner (1944), The anterior region also 
exhibits more activity as compared to the remainder of the section. 

This is the region of pre-stalk cells. 

Figt£e V. 

This is a section of a late migrating pseudoplasmodium which 
will shortly cease migrating and begin to culminate. Hie cells in 

- 17 - 

the anterior one-third of the section (pre-stalk region) are definitely 
aligned in a plane perpendicular to the longitudinal axis of the organ- 
ism.’ The cells in the poster!. or two-thirds of the organism (pre-spore 
region) are oriented randomly. A difference in the staining character- 
istics of the two regions is also evident, the pre-stalk region being 
slightly darker than the pre-spore region. 

FI gore VI . 

In the stage of development represented in this photomicrograph, 
the slime mold has ceased migrating and the aatero-posterlor axis 
assumes a vertical orientation with respect to the substrate. A con- 
figuration of cells resembling a crescent is seen in the apical portion 
of the section. In the entire organism, these cells fora an open-ended 
cylinder, the sides of which appear as two vertical lines of cells in 
the photograph. These form the sides of the crescent which on close 
examination can be seen to lack a connection across the top. The walls 
of the cylinder are one cell wide. These cells are horizontally alibi- 
ed and lay one atop the other. The level at which this cylinder occurs 
strongly suggests that it is concerned with the formation of the future 
sorophore sheath which will enclose the stalk of the organism. 

The cells Inside the cylinder are beginning to show vacuolization. 
They are round in shape and exhibit alkaline phosphatase activity to a 
lesser legree than the cells of the cylinder surrounding them. The 

- 18 - 

localization of the enzyme in these cells is not as diffuse as in 
the cylinder cells but rather appears to be limited to definite 
"spots" of activity. The semi-vacuolated state of these cells 
indicates that they are in the process of transforming into stalk 

The cells outside the cylinder have also become elongated, 
approaching the shape of the cylinder cells. They have assumed a 
horizontal orientation rather than the vertical orientation of the 
pre-stalk cells of the migrating pseudoplaamodium. Their location 
in the organism suggests that they are components of the pre-stalk 
region. The staining of these cells is lighter than that found in 
the cylinder cells. 

At the uppermost extreme of the section, the horizontally 
aligned cells above the cylinder merge imperceptibly with the 
rounded cells already described. These latter in turn enter the 
area between the walls of the cylinder and grade into the cell 
mass which is undergoing vacuolization. 

The remainder of the section is composed of the pre-spore 
nass* In this area there is no evidence of any high degree of 
alkaline phosphatase activity taking place. Activity appears to 
be at a minimum* The cells of this region show no elongation but 


retain their rounded configuration. Also, there is no continuation 
of the cylinder in this part of the organism, and in no portion of 
the slime mold has the sorophore sheath appeared as yet. 

Figure VIZ , 

This is a section of an individual more advanced in develop- 
ment than that in Figure VI, The sorophore sheath, i,e», the 
membrane which will enclose the stalk proper, is especially well 
defined. Its length, however, is limited to the region visible 
in the photograph. The vacuolated cells, which for the most part 
are surrounded by the sheath, extend below the lower limits of the 
sheath into the pre-spore mass, but as yet they do not reach to the 

Certain characteristics, indications of which could be seen 
in Figure VI, have now become more obvious. The horizontal elonga- 
tion of the cells outBide the sorophore sheath and the pronounced 
alkaline phosphatase activity in these cells is easily seen, Evident 
also is the lack of continuity across the upper end of the sorophore 
sheath. It appears that the cells at the apex of the section flow 
over the upper edges of the sheath into the area of the stalk itself. 
These are round cells which show only a slight degree of alkaline 
phosphatase activity. Progressing down the stalk region, increased 


vacuolization of the stalk colls is evident. At the lowest limit of 
the stalk, thie process is complete. Enzymatic activity is not 
evident in the completely vacuolated cells. In the surrounding 
pre-spore mass at the hase of the section, alkaline phosphatase 
activity is at a minimum. 

.te mi- 

In this stage of development , the pre-spore mss has begun to 
rise above the substrate. A band of heavy staining is seen in the 
center of the section which completely encircles the organism at 
this level. Its position suggests that it is the portion cf the 
pre-stalk region immediately adjacent to the upper limits of the 
pre-spore region. A very weak positive staining is evident in the 
pre-spore region. Above the band of heavy staining can again be 
seen the horizontally aligned cells which, as before, merge with 
the rounded cells at the apex of the section. Here, as in Figure 
YU, continuity of the eorophore sheath at its upper limits can not 
be observed. 

Examination of all the serial sections of this individual 
reveals that the stalk as well as the sorophore sheath now extend 
through the center of the organism to the substrate. A gradient 
of increasing vacuolization in the stalk cells is apparent starting 
from the rounded cells of the upper extreme to the completely 

> 21 ' 

vacuolated cells In contact with the substrate* A corresponding 
decrease in staining parallels this gradient. 

In this section the pre-spore mass has risen almost clear of 
the substrate. The entire length of stalk is visible and reveals 
the transition of the stalk cells from the sect -vacuolated type 
at the top of the stalk to the completely vacuolated cells at the 
bottom. Portions of the sorophore sheath can be seen in the upper 
and lower halves of the stalk. Here, ae in Figures VTI and VTTI, 
the horizontally aligned cells surround the upper portion of the 

A new trend exhibits itself in this section. An exceedingly 
dark-staining area is apparent in the lower half of the pre-stalk* 
region. It is much darker than any positive staining reaction seen 
thus far in the developmental cycle. The darkness of the staining 
suggests that a high degree of alkaline phosphatase activity occurs 
in this region. This highly active area is sharply demarcated from 
the lower pre-spore mass. The staining extends completely around 
the organism at this level and is separated from the etalk ares by 
the sorophore sheath. 

Ho sites of enzymatic activity were evident in the completely 


vacuolated cells of the lower stalk. The cells of the upper stalk. 

however, "being incompletely vacuolated, showed a alight degree of 
activity. A similar low degree of activity was found in the pre- 
spore mass. 

figure X. 

This is a section through a culminating individual with the 
pre-spore mass raised completely above the substrate. Many of the 
same features can be pointed out in this section as were present in 
figure IX. These are the high degree of enzymatic activity in the 
horizontally aligned cells of the pre-stalk region, the weakly 
active, serai-vacuolated cells of the upper stalk, the completely 
vacuolated lower stalk cells showing no activity, and the presence 
of the sorophore sheath. 

In addition, several new features are present for the first 
time. The cells of the nre-spore mass have begun to transform into 
mature spore cells. It is during culmination that the pre-spore myx- 
araoebae undergo this transformation (Bonner, 19^» Baper and Fennell, 
1952) . Isolated areas of enzymatic activity are seen in the sorogen. 
Furthermore , a second area of intense staining is found for the first 
time in the lower region of the sorogen. It extends downward, in close 
association with the stalk, into the basal disc region. The intensity 
of staining in this region is very much like that found in the pre- 
stalk area. Another curious similarity is the fact that here, too, 
the cells are aligned horizontally and elongated. 

- 23 - 

E&bhm M* 

In this individual, development has progressed further than 
in Figure X with the sorogen being raised appreciably higher above 
the substrate* Hie expanse and intensity of staining is quite 
striking. The pre-stalk region maintains its intense staining as 
before* An extension of this stained area however is seen to 
reach down into the spore mass and meets a similar upward extension 
from the darkly stained area at the base of the spore mass* This 
connecting portion is confined to approximately the width which 
is seen in the photomicrograph and does not extend out to the 
periphery of the spore mass. The area of intense staining at the 
base of the spore mss runs downward for some distance along the 
stalk. Hie complete absence of any alkaline phosphatase activity 
in the stalk cells is again noted. 

iiSKB m* 

This is a section through the spore cap of a mature individual. 
A portion of the stalk is seen imbedded in the spore mass. Actually, 
at this stage the stalk extends from the substrate to the uppermost 
tip of the spore cap. A minimum of activity is evident in the spore 
cells, while no activity was observed in the stalk cells. 


SpeclroDhotometr^e Analysis of Alkaline 
Phosphatase Activity 

The data derived from the spectrophotometric analyses of alkaline 
phosphatase activity are found in Table I and summarized in Table II* 

Table I 








0.493 . 



































Individual experimental data of spectrophotometrie 
analyses of alkaline phosphatase activity. Results 
are expressed in terms of jug P liberated/ jug tissue 
dry weight. These data represent the actual values 

x 102. 

Figure XHI illustrates the results graphically. From these data it 
may be stated thats 


1* When the enzymatic activity in the migrating pseudo- 
plaemodia is compared to that in the vegetative myxamoebae, no 
statistical difference is evident (P>0,05)*, 

2, A 113$ increase in alkaline phosphatase activity is ex- 
hibited by the young soroearps relative to the migrating pseudo- 
plasmodia and the vegetative myxamoebae. This difference is shown 
to be statistically significant (P< 0,001), 

3* Following this increase in activity in the young sorocarpa 
over the migrating pseudoplasmodia and vegetative myxamoebae, an 
abrupt and pronounced decrease in activity is found in the mature 
soroearps as compared to the young soroearps (P< 0,001), 

4, Analysis of enzymatic activity in the myxamoebae indicates 
that an increase in activity occurred relative to the nature soroearps 
(P< 0.001), 

^Student's "T* test was used to calculated probability 



Stage of 

Ho. of 

m P/j*g dry wt. 
Mean + S.D. 



0.57^ ♦ 0.053 




0.597 ♦ 0.046 

Young sorocarps 


1.380 ♦ 0.180 

Mature sorocarps 


0.147 + 0.040 

S ternary of data in Table I* Mean values of 
P& P liberated/ Mg dry tissue weight with 
their standard deviations. These data 
represent the actual values x 102. 



Bot#h histochemical and spectrophotoinetric techniques were used 
to study alkaline phosphatase activity in D. discoideum . The informa- 
tion gained by one method was complemented by the results shown by 
the other* The spectrophotometric analyses showed no difference in 
activity between the migrating pseudoplasmodia and the vegetative 
n.yxanoehae* However , a two— fold increase in activity occurred in 
the young sorocarps relative to both the myxaraoebae and migrating 
pseudoplasmodia* In the mature sorocarps, the activity dropped to 
a minimum* 

Based on the fact that the histochemical approach can be 

, • 

quantified to a certain degree (Gooori, 1952; Danielli, 1953), the 
same conclusions can be derived from the histochemical approach to 
this problem. During the course of development , only slight 
differences in intensity of staining were evident in the stages 
represented in Figures II through VIII. It is interesting to note 
that these differences, although slight, denoted increased activity 
only in the pre-stalk region* 3n Figure IX, a young sorocarp, an area 
of extremely dark staining became evident in the pre-stalk region* 

Thus, alkaline phosphatase activity increased in this region as 
compared to the activity in the pre-stalk area in the preceding 
stage* In succeeding stages, the activity in the same region not 
only maintained its intense staining characteristic but became more 


widespread, as is seen in Figures X and XT# Furthermore, enzymatic 
activity was observed in the region of the sorogen# The activity 
declined rapidly in the mature sorocarp and dropped to a rH mum. 

Not only do the results of these two methods of approach 
complement each other, but they are aB well correlated with other 
works done on D« discoideum # Bonner ( 1944 ) has shown that trans- 
formation of the pre-spore cells into mature spore cells first 
occurs at the upper periphery of the sorogen# Transformation then 
proceeds rapidly inward and downward through the sorogen untill 
all pre-spore cells are differentiated# An inspection of Figure 
XI which represents an individual approaching maturity, indicates 
that a somewhat similar pattern can be seen in the alkaline phosphatase 
localization# It is doubtful that this is a mere coincidence# The 
evidence would suggest that alkaline phosphatase activity is intimately 
concerned with the transformation of pre-spore cells to mature spore 
cells. The occurrence of alkaline phosphatase activity in both the 
pre-stalk area and sorogen of the young sorocarp is reflected in the 
spectro photometric data by a 113 ^ increase in activity over that 
found in the migrating pseudoplasmodia where somewhat pronounced 
activity, as revealed by the histochemical technique, was found only 
in the pre-stalk area# 


In Figure X f alkaline phosphatase activity use demonstrated 
at the lowor periphery of the sorogen as well as in the pre-stalk 
region* The cells in this region of the sorogen were aligned in 
nearly a transverse direction. Raper and Fennell (1952) also found 
such an orientation of the cells in this region in the same stage of 
development. Regarding this region, these authors believe that the 
cells therein ”... .exert an appreciable but continually decreasing 
role in raising the sorogen during culmination. “ The coincidence of 
such a postulate for the function of this region and the occurrence 
of high alkaline phosphatase activity in the same region would 
suggest that this enzyme may be concerned in Bone manner with this 

Histochemicel analyses have shown that alkaline phosphatase 
activity is most pronounced in the pre-stalk region. The pronounced 
activity in the pre-stalk region first occurred in the young migrating 
pseudoplasmodium and persisted during development until shortly before 
the formation of the nature eorocarp. In view of the fact that the 
pre-stalk region gives rise to the stalk, it would seem reasonable 
to suggest that alkaline phosphatase is concerned in some manner with 
certain mechanisms involved in the formation of the stalk. Other 
observations seem to substantiate this view. It was pointed out that 
in the culminating individual, the cells of the pre— stalk area assume 

• 30 - 

a horizontal or transverse orientation in addition to the fact that 
they become very densely stained. As these cells are followed upward 
over the open end of the sorophore sheath and into the sorophore 
proper, the horizontal cells merge with rounded, randomly oriented 
cells which are becoming vacuolated. These cells are then seen to 
enter the inside of the sorophore sheath and become completely 
vacuolated, cellulose-containing cells. A change in the enzyme 
staining characteristics is directly associated with this gradual 
change in cell architecture. In the horizontally aligned cells, a 
dark staining reaction occurs which decreases in intensity in the 
semi -vacuolated, round cells. A cessation of activity was found in 
the completely vacuolated cells of the stalk proper. 

By the use of numerous qualitative tests, Raper and Fennell 
(1952) were able to demonstrate quite conclusively that the main 
component of the sorophore was cellulose. According to them, the 
cellulose of the stalk cells, as well as the spore cells, is 
deposited intracellular ly. They further state that the first 
cellulose formed is deposited as a "....continuous film (sorophore 
sheath) adjacent to the surface of a layer or palisade of cells 
actively secreting cellulose synthesizing enzymes." An inspection 
of Figure III reveals not only two such palisades (the sides of the 

- 31 - 

d escribed cylinder) but also reveals that the cells of these 
palisades are elongated horizontally and are more pronounced in 
alkaline phosphatase activity than are the cells on either side 
of them* On the basis of relationships between these studies, it 
is tempting to suggest that alkaline phosphatase is concerned with 
the formation of the sorophore sheath* 

Gregg jgt al. (195*) and Gregg and Bronsweig ( 195* ) found an 
inverse relationship existing between protein content and amount 
of reducing substance (presumably carbohydrate)* This suggested 
that the synthesis of cellulose in the slime mold occurs at the 
expense of protein* As the amount of protein decreases during 
development, the quantity of reducing substance increases* In 
addition, alkaline phosphatase has been demonstrated in the present 
work in regions where cellulose is being formed* In the light of 
these facts, the following ppstulate is presented in an attempt 
to explain the manner in which protein is converted to cellulose* 

The limits of this postulate are defined not only by the comparative 
lack of biochemical data concerned with JD* discoideua . but also by 
the lack of more specific knowledge concerning the relationship of 
alkaline phosphatase to other enzymes and enzyme systems* 

It was shown by Gregg, e$. al. (195*) that the metabolism of 
proteinaceous substances increased as the development of the slime 

mold progressed. If this protein, or part of it, exists in the form 
of a nucleoprotein complex, then, presumably alkaline phosphatase 
is instrumental at this point in liberating the protein from the 
nucleic acid as was suggested by Eradfield (1951). This is not 
to say that alkaline phosphatase per se is the enzyme exercising 
such a function. In all likelihood, it would be part of an enzyme 
system acting in this capacity as was also suggested by Bradfield, 

The released protein could then enter an enzyme system for its con- 
version to carbohydrate. In view of the current concept involving 
phosphorylase and phosphatase actions for the biosynthesis of 
polysaccharides (Upmann, 19*1), it Is conceivable that this released 
protein after conversion to carbohydrate could be intracellularly 
deposited as the final product, cellulose, in the spore cells and 
stalk cells. In view of the "versatility" of action of alkaline 
phosphatase, it is further conceivable that the enzyme nay act again 
in this conversion by virtue of its phosphatase action. Pronounced 
alkaline phosphatase activity was noted during that period of develop- 
ment when stalk cells were being formed, as well as in that area of 
the young sorocarp where the transformation of pre-spore to mature 
spore cells containing a cellulose covering was occurring. 

As was indicated earlier, the sorophore sheath is thought to be 
formed next to cells secreting cellulose synthesizing enzymes (Paper 


and Fennell, 1952 ). J'oog and Wenger ( 1952 ) demonstrated the occurrence 
of high alkaline phosphatase activity and high concentrations of muco- 
polysaccharides in close association with each other* These authors 
went so far as to suggest that alkaline phosphatase itself was the 
polysaccharide moiety of the mucopolysaccharide complex* If so, the 
added evidence that alkaline phosphatase plays a "••••part in the 
carriage of organic substances across membrane barriers" (Yao, 1950), 
strongly suggests the possibility that alkaline phosphatase is also 
concerned with the extracellular deposition of cellulose to form the 
sorophore sheath* Whether it acts the role of ensyme in this function 
or whether it serves as the source of the polysaccharide is as yet 
not known* 

An attempt has been made to establish a series of correlations 
between the data and observations of this work with previously known 
facts concerning the biochemistry of Dictyosteliim * With additional 
work on this organism, now contemplated, it is felt that alkaline 
phosphatase will prove to play an important role in the developmental 
processes in DictYostelium discoideum as was suggested here* 



1. Techniques pertinent to the histochemical and epectrophoto- 
metrlc analysis of alkaline phosphatase activity in the developing 
slime mold, Pictyostellum dlscoideuBi . are described. 

2. The results of the histochemical techniques as applied to 
different stages in the development of the slime mold showed the 
following i sites of enzymatic activity in the vegetative myxamoebae 
were evidenced next to the nuclear membrane as r/ell as in the cyto- 
plasm itself. 

Enzyme activity in the migrating pseudoplasmodium, as well as 
in the preculmination stage, is limited for the most part to the 
pre— stalk area. Hiis activity is not too pronounced as compared to 
that which is demonstrated in the young sorocarp. In the young 
sorocarp, increased activity can be seen in the pre-stalk regions 
of the Individual slime molds. This increased activity is expressed 
in terms of not only darker staining characteristics but also in 
terms of a more wide spread distribution of the activity than was 
found in the previous two stages. 

3» The quantitative spectrophotometric analyses show that no 
appreciable change in activity occurs in the myxamoebae and the 

• 35 ' 

migrating pseudoplasmodia, relative to each other, A 113^ increase 
in activity is measured, however, in the young sorocarp stage 


relative to the previous two stages of development* An analysis 
of enzyme activity in the mature sorocarps reveals that the activity 
drops to a level below that found in the myxamoebae* 

4. The results of this work are discussed in the light of other 
studies on Piotyo3teliun disco idem in an attempt to describe the 
role which alkaline phosphatase may have in the development of this 



Figures III through XII are photomicrographs of serial 
sections of the slime mold, Dictvostelium discotdeum . Each of the 
sections is cut at 10 and they are presented in successive 
stages of development. Below each photomicrograph is a scale 
denoting 150 ju. 

The vegetative myxamoebae are shown in Figure II j (a) a 
photomicrograph, (b) a pen drawing. The myxamoebae average 5 /J 


in diameter. 


<3 ' 


Figure I 

Life cycle of the elirae mold, Dictvostelium 

38 - 

Figure Ila 

Photomicrograph of vegetative ayxaaoebae 

Figure lib 

Drawing of vegetative myxamoeb&e showing 
sites of alkaline phosphatase not too 
clearly defined in Figure Ila 


Figure HI 

The evolution of a pseudoplasmoditsn 
from an aggregation mass 


Figure IT 

An early or "young* pseudoplasmodium 

• 41 - 

Figure V 

A late or 

"old" pseudo plasraodium 


Figure VI 

A pBoudoplasoodium which has ceased migrat- 
ing and has righted itself 



Figure vri 

Ar individual in the preculrai nation 

« 44 « 

I 1 

Figure VIII 

Mid**pr ec ulmlnat ion stage succeeding 
the previous figure 


I 1 

Figure IX 

An individual in which the sorogen has 
been alsoat raised clear of the substrate 

I 1 

Figure X 

Young sorocarp in the culmination 

- 47 - 

Figure XI 

A nearly nature sorocarp in late culmin- 
ation stage 


I 1 

Figure XIT 
A mature sorocerp 

( X I0“ z ) 



Figure XIII 

Spectrophotooetric analysis of alkaline 
phosphatase activity* Graphic presentation 
of data in Table II* V»A* (vegetative 
amoebae); M*P* (migrating pseudoplaemodia); 
Y.S. (young sorocarps); and M.S, (nature 
sorocarps). These values are to be multi- 
■plied by 1CT 2 . 

- 50 ' 


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• • 

Jacoby, F. » 

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- 53 ' 

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Jerome Oldrich Krivanek was born December 27, 1924, at Chicago, 
Illinois. He attended elementary school and high school in that city 
and graduated from Farragut High School in 1941, He attended Hersl 
Junior College, Chicago, Illinois, from 1941 to 1943, 

After three years in the U. S. Army Air Force, he attended the 
University of Illinois, Urbana, Illinois, and received the Bachelor 
of Science degree in physiological zoology in February, 1948, 

He attended the Graduate School of the same university and 
received the Master of Science degree in physiological zoology in 
February, 1951. In September of that year, he enrolled in the Graduate 

k* • 

School of the University of Florida and has been engaged in studies and 
research leading to the degree of Doctor of Philosophy since that date. 
He has served as a research assistant for the academic year, 1952-1953, 
and as a graduate assistant for the remainder of the time. 

He is an associate member of the Society of the Sigma Xi. 

This dissertation was prepared under the direction of the 
chairman of the candidate's supervisory committee and has been 

approved by all members of the committee. It was submitted to the 
Dean of the College of Arts and Sciences and to the Graduate Council 
and was approved as partial fulfillment of the requirements for the 
degree of Doctor of Philosophy, 

June 6, 1955 

Dean, College of Arts and Sciences 

Dean, Graduate School